Automated developer for immuno-stained biological samples

ABSTRACT

Disclosed herein are systems and methods for the developing of immuno-stained biological samples. The systems disclosed herein are automated and are configured to control one or more steps of the developing procedure. Reagents may be added using automatic syringe dispensing. Reagent temperature, reagent stirring, and wash procedures are programmable and can be separately controlled for separate immuno-staining procedures that are performed at the same time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/693,492, filed Dec. 4, 2012, which is a divisional of U.S.application Ser. No. 12/644,839, filed Dec. 22, 2009, now U.S. Pat. No.8,337,754, the entire contents of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION Technical Field

The embodiments disclosed herein relate to automated systems forprocessing blots used in the field of molecular biology.

INTRODUCTION

Western blotting is one example of an immunostaining technique usedextensively for over 30 years in biology laboratories, in order todetect one or more target proteins in a sample. A schematic of theWestern blot procedure is shown in FIG. 1. First, the sample is loadedonto a gel and the proteins are electrophoretically separated in thegel, e.g., an SDS-PAGE gel, or a non-denaturing gel. (FIG. 1, step 1).The protein(s) within the gel are transferred, or blotted, onto filterpaper or other membrane, either by capillary forces, or byelectrophoresis, or blotted onto a piece of filter paper, e.g.,polyvinylidene difluoride (PVDF) or nitrocellulose (FIG. 1, step 2).Protein binding to the filter is based upon hydrophobic interactions, aswell as charged interactions between the membrane and protein. As such,the filter paper possesses non-specific protein binding properties inthat it generally binds all proteins substantially equally.

After transfer, the filter paper is treated, or blocked, to preventnon-specific binding of proteins at subsequent steps. Blocking ofnon-specific binding is achieved by placing the membrane in a dilutesolution of protein, such as bovine serum albumin or non-fat dry milk.The protein in the dilute solution attaches to the membrane in allplaces where the target proteins have not attached. Thus, when theantibody is added, there is no room on the membrane for it to attachother than on the binding sites of the specific target protein. Thisreduces “noise” in the final product of the Western blot, leading toclearer results, and eliminates false positives.

The detection of the target protein(s) is achieved either in a one-stepor two-step process. In the two-step process, the blocked filter istreated with a primary antibody specific to the target protein, followedby treatment with a secondary antibody specific for the primaryantibody, and which includes a detectable moiety. A dilute solution ofprimary antibody (generally between 0.5 and 5 μg/mL) is incubated withthe membrane under gentle agitation. The antibody solution and themembrane are incubated together for anywhere from 30 minutes toovernight. It can also be incubated at different temperatures, withwarmer temperatures being associated with more binding, both specific(to the target protein, the “signal”) and non-specific (“noise”).

The membrane is rinsed or washed to remove unbound primary antibody, andthen incubated with a secondary antibody specific for the primaryantibody and that contains a detectable moiety, which can be detected,as an indicator of the presence and/or amount of target proteins presentin the original gel. The secondary antibody is incubated with themembrane for a period of time, with gentle agitation.

Alternatively, in the one-step process, the blocked membrane can beincubated with a primary antibody that contains a detectable moiety,thereby eliminating the necessity for a secondary antibody.

Treatment of the blot with the primary antibody, and, if required,secondary antibody and developing solution to visualize the detectablemoiety, requires several steps of addition, incubation and washing,spread out over several hours. Automation of the Western blotdevelopment steps would advantageously reduce time and labor forprocessing samples. However, automation of Western blotting presentsunique challenges, due to the nature of the reagents used. Specifically,proteins such as antibodies are susceptible to degradation and aretherefore generally kept in a cold solution until they are ready foruse. Further, antibodies are generally expensive, and, in many cases arein limited supply. Thus, it is desirable to avoid using a larger volumeof solution, requiring larger volumes of antibody, in developing aWestern blot. There is thus a need for a system that can reduce the needfor manual manipulation of the Western blot, and that minimizes reagentwaste.

SUMMARY OF THE INVENTION

In one embodiment, the invention comprises an automated developer forimmuno-stained biological samples. The automated developer comprises aplatform with a surface configured to receive an incubation box, a towercoupled to the platform surface, and an incubation box comprising ahousing and a lid. The incubation box comprises a drain port and atleast one entry port positioned to allow entry of a reagent into theinterior of the incubation box. At least one syringe holder is provided,coupled to the tower and configured to receive a syringe, the syringeconfigured to hold a reagent within the syringe barrel, wherein thesyringe holder is positioned to hold the syringe relative to the entryport in order to allow delivery of the reagent from within the syringe,through the entry port, into the incubation box. Also provided is atemperature control device configured to control the temperature of thereagent within the syringe, a motorized syringe pusher configured tomechanically force the reagent from within the syringe barrel, throughthe entry port into the incubation box, and a stirrer configured toagitate the reagent within the syringe. A processor is configured tocontrol one or more of the motorized syringe pusher, the stirrer, andthe temperature control device.

In another embodiment, an automated method of processing animmuno-stained biological sample comprises providing at least a primaryantibody in a syringe, placing an undeveloped sample into an incubationbox, contacting the undeveloped sample with the primary antibody byautomatically activating a first motorized pusher to mechanically forcethe primary antibody from within the syringe barrel into the incubationbox, automatically removing the primary antibody from within theincubation box after the contacting step, automatically pumping a washbuffer into the incubation box, and automatically removing the washbuffer from the incubation box.

In another embodiment, an automated developer for immuno-stainedbiological samples comprises a processor controlled rocking platform, aprocessor controlled syringe pusher and a processor controlled bufferpump. The processor is configured to control addition of antibodyreagent to an incubation box by controlling the syringe pusher, and isconfigured to control addition of buffer the incubation box bycontrolling the buffer pump. The processor may be coupled to a userinterface for programming operational timing of the syringe pusher andbuffer pump.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the steps of a “two-step” Western blotting approach.

FIG. 2 is a perspective view of one embodiment of an automated blotprocessor.

FIG. 3A is a perspective view of one embodiment of an incubation box,and an incubation box lid.

FIG. 3B is a perspective view of the incubation box lid shown in FIG.4A.

FIG. 3C is a perspective view of the incubation box shown in FIG. 4A.

FIG. 3D is a planar view of one corner of an incubation box containing amembrane, and showing a drain channel.

FIG. 4A is a sectional view of a syringe housing and syringes of oneembodiment of the system disclosed herein.

FIG. 4B is a cutaway view of the syringe housing shown in FIG. 4A,showing the syringe housing, syringe, and magnetic stirrer of oneembodiment of the system disclosed herein.

FIG. 5A is a perspective view of one embodiment of a syringe pushingmechanism.

FIG. 5B illustrates motion of the syringe pusher within a guide channelof the automated developer.

FIG. 6 is a block diagram of the components of one embodiment of anautomated developer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The embodiments disclosed herein relate to systems and methods forreducing the amount of manual manipulation required for blottingtechniques used in molecular biology, while preserving reagents andminimizing waste.

Embodiments will now be described with reference to the accompanyingFigures, wherein like numerals refer to like elements throughout. Theterminology used in the description presented herein is not intended tobe interpreted in any limited or restrictive manner, simply because itis being utilized in conjunction with a detailed description of certainspecific embodiments disclosed herein. Furthermore, embodimentsdisclosed herein may include several novel features, no single one ofwhich is solely responsible for its desirable attributes or which isessential to the embodiments herein described.

Referring now to FIG. 2, a perspective view of an automated developer100 according to one embodiment is shown. The automated developer 100includes a platform 181 configured to receive one or more incubationboxes 330. In some embodiments, the incubation box 330 can be integralto the platform 181. In other embodiments, the incubation box 330 isseparate from and can be removed from and replaced onto the platform181. In some embodiments, the platform 181 is configured to agitate theincubation box. For example, in some embodiments, the platform 181 canbe a rocking platform configured to tilt the incubation box 330 and itscontents to various angles. This can be accomplished in a variety ofmanners, such as risers on the underside of the platform that arecoupled to rotating cam shafts, eccentrically mounted bearings, or thelike. A motor can turn the cam shafts, producing the rocking motion. Insome embodiments, the platform 181 is designed to tilt the contents ofthe incubation box 330 in more than one plane. In some embodiments, thetilt angle and the speed of the rocking platform can be adjusted. Insome embodiments, the tilt and speed parameters for the rocking platformcan be adjusted manually. In some embodiments, the tilt and speedparameters of the platform can be controlled by a processor (designated620 in FIG. 6) within the automated developer 100. In some embodiments,the processor includes a display and a user input interface, such as atouch screen 210. It will be appreciated that keypads, LED indicators,etc. could alternatively be used, although the touch screen display/userinput interface is user friendly and flexible to allow for programmingthe actions of the developer as described further below.

In some embodiments, the surface 182 of the platform 181 includes adepression 160, configured to fit the incubation box 330 within thedepression 160 to hold the incubation box 330 in place. In someembodiments, the incubation box can be configured to be locked onto thesurface 182 of the platform, e.g., by a snap-lock mechanism or the like.The embodiment of FIG. 2 illustrates a platform with locations foraccepting four incubation boxes 330.

In some embodiments, the automated blot processor includes a tower 110attached to the platform surface 182. Tower 110 is configured to movewith the platform 181, e.g., a rocking platform. The tower 110 caninclude one or more syringe holders 220, as described further below. Thetower 110 can also include one or more channels 120, which hold pusherarms 250, as described below.

Turning to FIG. 3A, a perspective view of an exemplary incubation box330 is shown. The incubation box includes an incubation box housing anda lid. Perspective views of the incubation box housing and the lid areshown in FIGS. 3B and 3C, respectively. The incubation box housing canbe configured to hold a filter or membrane 370, e.g., for a blot, andreagents, solutions, and the like, for incubation of the filter ormembrane 370 during various stages of the blotting procedure.Preferably, the incubation box 330 is rectangular, although the skilledartisan will recognize that the shape of the incubation box 330 can bealtered, so long as it is suitable for holding a membrane 370 andregents, solutions and the like. In some embodiments, the bottom innersurface of the blot housing includes a drain channel 360. In embodimentswherein the incubation box is rectangular, a drain channel can belocated in one or more corners of the bottom inner surface of the blothousing.

The incubation box lid is configured to fit on top of the incubation boxhousing. The lid can be removably attached to the box housing, e.g.,through a hinge and snap lock 350, or the like, that allows open andclosing of the lid onto the incubation box housing. In some embodiments,the lid is not attached or coupled to the box housing but rathercomprises a lip that is complementary to and fits around the outside ofthe box housing, keeping the lid in place on the box housing similar toa typical shoe box.

In some embodiments, the incubation box 330 can be disposable, andconfigured for single use. In some embodiments, the incubation box 330can be made of reusable material.

As shown in FIG. 3B, the incubation box lid can have one or more entryports, (e.g., one, two, three, four, or more entry ports), eachconfigured to allow entry of reagents and solutions from a reagentsource or a syringe into the interior of the incubation box housing. InFIG. 3B, the lid includes primary antibody entry port 380, a secondaryantibody entry port 390, and a wash buffer entry port 400.

As shown in FIG. 3D, the incubation box housing 180 includes a drainfeature 401, which allows for removal of reagents and buffers fromwithin the incubation box housing. In some embodiments, the drainfeature 361 includes a drain channel 360 leading into the waste port361. In some embodiments, the waste port 361 is coupled to drain tubing190. Various types of tubing known to those skilled in the art can beused in the embodiments disclosed herein including, but not limited to,bioperene thermoplastic elastomer tubing (Watson Marlow Bredel Products,Wilmington, Mass.), CHEM-DURANCE™ chemical pump tubing (COLE PARMER®,Vernon Hills, Ill.), and the like. In some embodiments, the tubing issecured to the waste port 361 by a tubing clamp 402. In someembodiments, the tubing is maintained in place in the waste port byfrictional forces. In some embodiments, the tubing is maintained inplace by a combination of, for example, a clamp and frictional forces.As shown also in FIG. 2, the drain tubing 190 enters the waste port fromabove and extends down to the drain channel located just off one bottomcorner of the incubation box. The tubing 190 can lead to a wastecontainer. In some embodiments, the tubing secured to the waste port 361is coupled to a pump, such as a peristaltic pump, a piston pump, ahydraulic pump, or the like, via drain tubing 190. In preferredembodiments, the pump is a peristaltic pump. The pump can be used tocause the flow of reagents and solutions out the waste port 361, up intothe drain tubing 190, and out of the incubation box.

It has been found advantageous to form the waste port 361 where thedrain tube 190 attaches to the incubation box as an external feature ofthe rectangular perimeter of the remainder of the incubation box.Although it is possible to construct the system such that the drain tubemerely enters the main portion of the incubation box from the topthrough the lid, the suction of the drain tube can pull the blot paperagainst the end of the drain tubing 190 and prevent good drainage fromthe box. In the embodiment of FIGS. 2 and 3, the blot paper cannot comeinto contact with the end of the drain tube 190. Furthermore, the drainchannel 360 can include a downward draft angle, which advantageouslyallows for solutions and reagents to flow towards the waste port 361where the end of the drain tubing 190 is located, while at the same timeavoiding flotation of the blot toward the waste port 361 because thecorner of the blot paper will rest in the corner of the main portion ofthe incubation box, and will not come into contact with the end of thedrain tubing 190.

In some embodiments, the incubation box lid contains an entry port for abuffer source. In some embodiments, the buffer source can be coupled tothe entry port via tubing. In some embodiments, the tubing can beconnected to a pump, configured to force a buffer or solution throughthe tubing into the box housing. For example, in some embodiments, washbuffers, and the like used in. Western blots can be delivered to theentry port via tubing 200 that is connected, e.g., via a peristalticpump.

Turning now to FIG. 2, FIG. 3 and FIG. 4, in some embodiments, thereagent entry port(s) 380, 390 of the incubation box lid are positionedbelow syringes 230, 240, which are secured within syringe holders 130,140 in a syringe housing 220 of the automated developer 100. Forexample, in some embodiments, a syringe 230, 240 can be filled with areagent, and the entry ports 380, 390 can be positioned in line witheach syringe tip 300 from which the reagent exits the syringe barrel270, such that the reagent exits the syringe and passes through theentry port 380, 390 into the interior of the incubation box 330. Asdescribed further below, the contents of the syringe can beautomatically added to the incubation box at the appropriate time bycontrolling a syringe pusher arm 250.

Automated delivery of reagents with the syringes shown in FIGS. 2 and 4has a large advantage over the use of pump reagent delivery. Reagents,and in particular antibodies, are generally expensive, and in limitedsupply. The use of syringes 230, 240 as a reagent source advantageouslyeliminates dead volume, thereby reducing waste of valuable reagents.When the syringe plunger 260 is driven downward into the syringe barrel231, the reagent is expelled from the syringe body with little to nowaste. Those skilled in the art will appreciate that any syringesuitable for the purposes of the methods described herein can be used.In some embodiments, the syringe can be a 1 cc syringe, a 5 cc syringe,a 10 cc syringe, a 20 cc syringe, a 25 cc syringe, or the like.

FIG. 4 shows an example of a syringe housing 220 in more detail. FIG. 4Ais plan view of the syringe housing. Temperature sensitive reagents thatare susceptible to degradation are preferably kept at coolertemperatures until use. The features of the syringe housing describedherein overcome the challenge of having to manually thaw and addreagents such as antibodies to an immuno-staining procedure such as aWestern blot. In some embodiments, the syringe housing includes atemperature control feature, that cools (or possibly heats) the reagentsas required. This feature advantageously eliminates the requirement ofthawing and manually adding reagents, such as antibodies, just prior touse. Due to the fact that the reagents can be kept cool and do not needto be thawed and added throughout the procedure, the automation of thesequential incubation and washing steps of the procedure is enhanced.

Accordingly, in some embodiments, the syringe housing can also include atemperature control device positioned relative to the syringe to be ableto control the temperature of the reagent within the syringe. In someembodiments, the temperature control device is a cooler, such as aventilated Peltier junction thermoelectric cooler, a fan, and arefrigerated jacket that surrounds the syringe, or the like. In someembodiments, the temperature control device can also heat the reagents.In some embodiments, the temperature control device can be controlled bythe processor. In some embodiments, the syringe housing includes atemperature control device that keeps the reagents within the syringes230, 240, at −10° C., −5° C., 0° C., 4° C., 10° C., 25° C., or the like,during the developing process. A temperature sensor (630, FIG. 6) suchas a thermocouple or thermistor is advantageously provided for feedbackto the control processor so that programmable temperature regulation canbe performed.

Shown in FIG. 4A is a syringe housing that includes a fan 150 andpeltier cooler 320, that function to keep the reagents, e.g., antibodiesand the like, cool during the blot process, in order to avoiddegradation of the reagents. For example, in some embodiments, the fanand peltier cooler can be configured to keep reagents within thesyringes 230, 240 between about 0° C. to about 10° C.

In some embodiments, the syringe housing 220 can include two openings130, 140, to hold two syringes 230, 240, configured to house twodifferent reagents, e.g., a primary antibody and a secondary antibody,respectively. The skilled artisan will appreciate that the syringehousings can be configured to hold one, two, three, four, five, six,seven, eight, nine, ten, or more syringes, depending upon theapplication. For example, in some embodiments, a primary antibody with adetectable label incorporated therein can be used in a Western blot,thereby eliminating the need for a secondary antibody. Accordingly, insome embodiments, the syringe housing 220 of the automated blotprocessor 100 can include one syringe opening 130, configured to receiveone syringe 230.

FIG. 4B is a cutaway view of the syringe housing shown in FIG. 4A. Apeltier cooler 320 can be positioned between the fan 150 and the syringe240. The syringe fits within the syringe opening 130 and is insertedinto a temperature controlled jacket 408. The Peltier cooler 320 drawsheat from this jacket to heat sink 314 which is cooled by the fan 150.The plunger 260 is shown within the syringe barrel 231. In theembodiment of FIGS. 2 and 4, a single jacket 408, Peltier cooler 320,and fan 150 are used to control the temperature of two syringes. It willbe appreciated that separate temperature control for each of multiplesyringes can also be performed.

In some embodiments, a magnetic disc 290 or stirrer can be disposedwithin the reagent source, e.g., a syringe barrel 231, in order toagitate the reagent. As shown in FIG. 3B, the syringe housing 220 caninclude device for introducing a magnetic field to rotate or move themagnetic disc or stirrer 290, such as a solenoid coil 310, anelectromagnet, a permanent magnet, or the like. In preferredembodiments, the device is a magnetic disc 290 that can lay flat in thebottom of the syringe barrel 231 when the solution is not beingagitated, displacing as much reagent as possible out of the syringe whenthe plunger is depressed. In some embodiments, the processor is used tocontrol the magnetic disc 290 or stirrer at pre-programmed timeintervals via an electromagnet or solenoid coil 310, although it will beappreciated that in some embodiments the stirrer can be rotatedcontinuously. Control of the stirring at pre-determined time intervalsby the processor is advantageous in that it minimizes the heat generatedby the stirrer, and thus reduces the risk of overheating reagents thatare at risk of becoming denatured in excess heat. By way of example, thesolenoid coil 310 can be energized with a biphasic voltage waveform withan amplitude and frequency adjusted to achieve optimal rotation of thedisc or stirrer 290. Other means for stirring or agitating the reagentcan be used, including but not limited to a physical stirrer, a deviceto deliver ultrasonic vibrations to the reagent source, or the like. Aswith the magnetic stirrer, such the alternative means for stirring canalso be controlled by the processor.

Turning back to FIG. 2, in some embodiments, the automated blotprocessor 100 can include a motorized pusher that is configured to pushthe syringe plunger 260 down into the syringe barrel 231, expelling thecontents in the barrel out of the syringe 230, 240, and through theentry port 380, 390. In some embodiments, the pusher can comprise an arm250 that extends out of a channel 120 in the tower and is aligned alongthe travel path of the syringe plunger 260. The arm is configured to beautomatically moved up or down along the height of the tower 110,thereby forcing the plunger 260 into syringe barrel 231 via downwardtravel at the appropriate programmed times. In some embodiments, thechannel is angled the end of the channel that is distal to the platform(the “top” of the channel). The angle at the top of the channel 120accommodates the pusher arm 250 in a position that is not directly abovethe syringe while the syringe is being loaded or unloaded into thesyringe openings 130, 140.

In some embodiments, the pusher arm 250 is coupled to an internallythreaded riser attached to an externally threaded shaft driven by amotor. This is illustrated in FIGS. 5A and 5B, which shows oneembodiment for mechanically controlling a pusher arm 250. In thisembodiment, an internally threaded riser 500 and externally threadedshaft 510 can be located within the tower, with the pusher arm 250extending out of the channel 120. The motor 520 can rotate theexternally threaded shaft 510 within the internally threaded riser 500.The pusher arm is forced against the walls defining the channel 120,which prevents rotation of the riser, and instead causes the riser tomove up and down the shaft, depending upon which way the motor turns theshaft.

In some embodiments, the processor controls the motor that moves thepusher arm 250 up and down the channel 120 of the tower 110. As the armmoves down the channel 120 in the tower 110 it forces the syringeplunger 260 into the syringe barrel 231. The processor may then reversethe direction of the motor, in order to move the pusher arm 250 back upthe channel 120 after the contents of the syringe 230, 240 have beenexpelled from the syringe barrel 231 into the incubation box housing180. In some embodiments, when the arm 250 travels back up the channel1210, rotation of the motorized shaft automatically moves the pusher arminto the angled portion of the channel, so that the syringe 230, 240 canbe easily removed. This is illustrated in FIG. 5B, where motor rotationpushes the arm 250 against the left channel wall when the riser ismoving up, and against the right channel wall when the riser is movingdown. When the arm 250 moves up into the upper angled portion of thechannel 120, motor rotation will keep the arm 250 against the left wall,and move the arm away from being directly above the syringe. When thearm is moved down again, motor rotation first pushes the arm 250 againstthe right channel wall, and then the arm moves down into the lowerportion of the channel 120.

In some embodiments, the automated developer is connected to one or morebuffer sources via tubing 200. In some embodiments, a pump, such as aperistaltic pump controls the movement of buffer from the buffer source,into the tubing. In some embodiments, the tubing is attached using anyconnector known in the art and suitable for the embodiments disclosedherein, e.g., a luer lock, to an entry port on the incubation box lid.

FIG. 6 is a block diagram of the components of the developer of FIG. 2illustrating the automation control paths in this embodiment. Thedeveloper may include some control and processing circuitry 620 whichmay be programmable through a user input device. The control circuitry620 controls a motor driving the rocking platform. Also, the controlcircuitry can separately control four reagent and buffer input/drainstations as are shown in FIG. 2. With this configuration, separatereagent and buffer addition and removal schedules can be programmed intothe developer. In some cases, it is convenient to program the processingcycles for different incubation boxes so that they end at the same time,even if they have different schedules for adding reagents. Tosynchronize the completion time, it is possible to extend, for example,one or more wash periods for those incubation boxes that have earlyreagent addition and removal schedules. Generally, one or more of thewash or buffer cycles can be extended without affecting the immuno-staindevelopment. This feature frees the scientist and technician from alarge amount of otherwise manual monitoring and development steps whichquickly expand to a burdensome number when different immuno-staindevelopment procedures have some different desired incubation times forsome of the steps.

It will be appreciated that the processor element of the automateddeveloper 100 may be integral to the housing of the developer itself, orall or part of the processing and control circuits can be separate fromthe developer itself. In some embodiments, the processor can be aspecialized microcontroller which is designed specifically forcontrolling the elements of the automated developer device.Alternatively, the processor can be a standard personal computer devicesuch as an Intel processor-based PC running an off the shelf operatingsystem such as Windows, Linux, MacOS, or the like. As used herein, theterm “processor” generally refers to one or more logic and controlcircuits which are connected to the automated developer 100 to controlthe operation of various components of the automated developer asdescribed herein. In some embodiments, the processor can include directhardware interface such as a USB port, an RS232 interface, and IPnetwork interface (wired or wireless), or some other type of connection,to load software to control the components and functions of theautomated blot processor. In some embodiments, the processor isintegrated into the automated developer, which then interfaces with atouch-screen user interface 210, that enables the user to set theparameters for automated control of the different components of theautomated developer.

In some embodiments, the processor can include software that allows theuser to enter the timing and parameters for controlling one or morecomponents of the developer 100, such as the motorized pusher, therocking platform, the temperature control, the peristaltic pump(s), andthe like. In some embodiments, the software allows the user to programthe developer to complete a Western blotting procedure, includingcontrolling the following: the addition of reagents, such as blockingbuffer, wash buffer, primary and secondary antibodies, and the like tothe incubation box at predetermined times; the rocking of the platform;the drainage of buffers and reagents from within the incubation box;stirring of reagents within the syringes; controlling the temperature ofthe reagents; and the like. In some embodiments, the processor can allowfor automated collection of “run data” including, for example,temperature and volume measurements, reagent volume and incubation time,operator identity, date and time, etc.

In embodiments wherein the automated developer includes more than onesyringe holder 220, and more than one incubation box 330, the processorcan be set to add and remove reagents to each different incubation box330, enabling individual Western blots that require different incubationtimes with different reagents to be processed at the same time.

Some embodiments provide a method of processing a Western blot, usingthe automated developer 100 described herein. In some embodiments, auser will manually aspirate reagents such as a primary antibody, into asyringe, and place the syringe in the syringe holder. In someembodiments, the Western blot procedure includes an incubation step witha secondary antibody if the primary antibody is not labeled with adetectable label. In such embodiments, the user can aspirate thesecondary antibody reagent into a second syringe, and place the secondsyringe within the syringe holder.

The user can place one end of a buffer line into a buffer source. Insome embodiments, there is more than one buffer line, each connected toa different buffer source. For example, the user can place a firstbuffer line into a first buffer source containing blocking buffer and asecond buffer line into a second buffer source containing wash buffer.

The user can place a filter onto which proteins have been transferredinto the incubation box housing, and placing the housing lid onto theincubation box. The user can then set the automated developer to performthe steps of: contacting the undeveloped blot in the incubation box withthe primary antibody by activating the first motorized pusher tomechanically force the primary antibody from within the first syringe;agitating, e.g., by rocking the incubation box for a period of time;draining the primary antibody from the incubation box; pumping a washbuffer from a buffer source into the interior of the incubation box;rocking the incubation box for a period of time; removing the washbuffer from the incubation box; contacting the blot with the secondaryantibody by activating the second motorized pusher to mechanically forcethe secondary antibody from within the second syringe; and removing thesecondary antibody from within the incubation box.

The above described blot processor has many significant advantages.Automating the dispensing of antibodies from syringes eliminates asignificant amount of waste that would occur if the antibodies werepumped into the incubation box with a pump and tubing system. Antibodysolutions in the syringes can be temperature controlled andautomatically stirred. Separate control of multiple developmentprocesses simultaneously is also provided.

It will further be appreciated that blots are not the only biologicalsamples that can be developed using the above described automateddeveloper. It is often desirable to stain tissue slices with antibodiesto produce visual indications of the presence or absence of differenttypes of proteins, DNA or other biological molecules in a given sampleof tissue. This staining procedure also involves the application ofbuffers and antibodies to the tissue samples for selected incubationtimes and these procedures can also be automated with the automateddeveloper described herein. In some cases, the tissue samples arerelatively small, and the incubation box described above can besegmented with mesh walls that allow the solutions to pass through butmaintain separate tissue samples in separate portions of the incubationbox.

The above-described embodiments have been provided by way of example,and the present invention is not limited to these examples. Multiplevariations and modifications to the disclosed embodiments will occur, tothe extent not mutually exclusive, to those skilled in the art uponconsideration of the foregoing description. Additionally, othercombinations, omissions, substitutions and modifications will beapparent to the skilled artisan in view of the disclosure herein.Accordingly, the present invention is not intended to be limited by thedisclosed embodiments.

What is claimed is:
 1. An automated developer for immuno-stainedbiological samples, comprising: a processor controlled rocking platform;a processor controlled syringe pusher; a processor controlled bufferpump; wherein the processor is configured to control addition ofantibody reagent to an incubation box by controlling the syringe pusher,and is configured to control addition of buffer the incubation box bycontrolling the buffer pump.
 2. The automated developer of claim 1,wherein the processor is coupled to a user interface for programmingoperational timing of the syringe pusher and buffer pump.
 3. Theautomated developer of claim 1 further comprising a syringe housing. 4.The automated developer of claim 3 further comprising a processorcontrolled temperature control feature, wherein the processor isconfigured to control the temperature of a reagent.
 5. The automateddeveloper of claim 4, wherein the temperature control feature isconfigured to control the temperature of a reagent within the syringehousing.
 6. The automated developer of claim 3 further comprising aprocessor controlled stirrer, wherein the processor is configured tocontrol the speed and timing of the processor controlled stirrer.
 7. Theautomated developer of claim 6, wherein the processor controlled stirrercomprises a magnetic stirrer and a magnetic field generator, wherein themagnetic field generator is located in the syringe housing, and isconfigured to control the magnetic stirrer when the magnetic stirrer islocated within a syringe located in the syringe housing.
 8. Theautomated developer of claim 1, wherein the incubation box is connectedto the rocking platform, and wherein the processor is configured toagitate the incubation box by controlling the rocking of the rockingplatform.
 9. The automated developer of claim 1, wherein the processoris configured to control the duration and the intensity of the rockingof the rocking platform.
 10. The automated developer of claim 1, whereinthe incubation box comprises a waste port in fluid communication with aprocessor controlled pump, wherein the processor is configured tocontrol flow out of the incubation box and through the waste port.